Nikkomycin analogs

ABSTRACT

Compounds of the formula ##STR1## wherein R 3 , R 4 , J, K, Z, and Het are as set forth herein are described. 
     The compounds of formula I are useful as agents in the treatment of fungal infections.

This is a division of application Ser. No. 08/253,640, filed Jun. 3,1994 now U.S. Pat. No. 5,461,055, which in turn is a continuation ofSer. No. 900,712, filed Jun. 18, 1992 now U.S. Pat. No. 5,346,898, whichin turn is a continuation-in-part of Ser. No. 07/747,554 which was filedAug. 20, 1991, now abandoned.

SUMMARY OF THE INVENTION

The invention relates to compounds of the formula ##STR2## orpharmaceutically acceptable salts thereof, wherein Het is ##STR3## R isH, COOH; C₁ -C₁₂ alkyl; CHO; CN; CH₂ OH; or CONH₂ ;

wherein R₃ is ##STR4## wherein R₈ is ##STR5## R₂ is H; OH; F; C₁ -C₆alkoxy; alkyl; SH; S-alkyl; or SO₂ -alkyl;

R₄ is H; a natural amino acid attached by a peptide bond; or ametabolizable group;

R_(x) is C₁ -C₁₂ alkyl;

J is OH, H, Br, Cl, or F;

K is OH, H, Br, Cl, or F;

X and Y are the same or different and are independently selected fromthe group consisting of H; OH; O--C₁ -C₁₄ alkyl; F; Cl; Br; I; NO₂, andalkyl;

Z is R₅ NR₆ ; ##STR6## wherein Z' is R₅ NR₆, R₅ is H, a saturated orunsaturated C₆ -C₁₈ aliphatic side chain; or a hydroxylated C₆ -C₁₈aliphatic side chain;

R₆ is H; OH; benzyl; substituted benzyl; O-benzyl; O-aryl; O--C₄ -C₁₄alkyl; C₁ -C₁₂ alkyl; phenyl; substituted phenyl; or CO--R₇ ; and

R₇ is H, C₁ -C₁₆ alkyl, aryl or alkylaryl; and

n is an integer from 0 to 16.

Preferred are compounds of formula I wherein Het is uracil.

Also preferred are compounds of formula I: wherein R₄ is H.

Preferred are compounds of formula I where J and K are both OH.

Preferred are compounds of formula I wherein R₅ is a C₁₀ -C₁₈ saturatedaliphatic side chain:

Most preferred are compounds of formula I wherein R₅ is astraight-chained --(CH₂)₁₁ CH₃.

Preferred are compounds of formula I wherein the stereochemistry at the2" position is S according to Cahn, Ingoid, Prelog rules ofstereochemistry or L according to the natural stereochemistry of aminoacids. Also encompassed by formula I are compounds of the oppositestereochemistry, that is, wherein 2" position has the R or Dconfiguration.

Formula I is reproduced just below to show where the 2" and 1" positionsare. ##STR7##

Alkyl denotes straight or branched hydrocarbon chains, which containfrom 1 to 20 carbon atoms. Representative examples include methyl,ethyl, propyl, decyl, dodecyl and the like. Alternatively, the number ofcarbon atoms in a particular alkyl may be specified. For example, C₁ -C₆alkyl refers to an alkyl which may have one to six carbon atoms.

Alkoxy denotes O-alkyl wherein alkyl is as described above.

As used herein the term UR denotes uracil. The structural formula forthe uracil moiety is ##STR8##

The term UPOC denotes uracil polyoxin C. The structural formula for UPOCis ##STR9##

The term natural amino acid denotes the following amino acids; glycineand the following which have an L-configuration: valine, leucine,isoleucine, serine, aspartic acid, asparagine, glutamic acid, histidine,alanine, proline, phenylalanine, tryptophan, methionine, threonine,cysteine, tyrosine, glutamine, lysine and arginine which are alsoreferred to respectively by the following abbreviations:

Gly, Val, Leu, lie, Ser, Asp, Asn, Glu, His, Ala, Pro, Phe, Trp, Met,Thr, Cys, Tyr, Gln, Lys and Arg.

The term metabolizable group denotes any group which under metabolizableconditions (that is, when subjected to metabolic enzymes and the like)will be cleaved off. An example is ##STR10##

The term saturated aliphatic side chain denotes a straight or branchedsaturated side chain containing only carbons and hydrogen. The saturatedaliphatic side chain may contain up to 20 carbon atoms. Alternatively,the number of carbon atoms in an saturated aliphatic side chain may bespecified. For example, a saturated C₆ -C₁₈ aliphatic side chain denotesa saturated aliphatic side chain having from 6 to 18 carbon atoms. Thesaturated aliphatic side chain may contain within the chain up to threeof any combination of the heteroatoms O, S, or N, with the proviso thatsuch heteroatoms cannot be adjacent to each other in the chain. Thesaturated aliphatic side chain may also have a carboxyalkylfunctionality.

The term unsaturated aliphatic side chain denotes a straight or branchedaliphatic side chain containing up to three double or triple bonds orany combination thereof. For example an unsaturated aliphatic side chainmay contain three double bonds, two double bonds and one triple bond,one double bond and two triple bonds, or three triple bonds. Theunsaturated aliphatic side chain may contain up to 20 carbon atoms.Alternatively, the number of carbon atoms in an unsaturated aliphaticside chain may be specified. For example, a unsaturated C₆ -C₁₈aliphatic side chain denotes an unsaturated aliphatic side chain havingfrom 6 to 18 carbon atoms. The unsaturated aliphatic side chain maycontain within the chain up to three of any combination of theheteroatoms O, S, or N, with the proviso that such heteroatoms cannot beadjacent to each other in the chain. The unsaturated aliphatic sidechain may also have a carboxyalkyl functionality.

A hydroxylated aliphatic side chain denotes a saturated or unsaturated,straight or branched aliphatic side chain as described above, wherein upto 3 hydrogens are replaced by OH. The hydroxylated aliphatic side chainmay contain up to 20 carbon atoms. Alternatively, the number of carbonatoms in an hydroxylated aliphatic side chain may be specified. Forexample, a hydroxylated C₆ -C₁₈ aliphatic side chain denotes anhydroxylated aliphatic side chain having from 6 to 18 carbon atoms. Thehydroxylated aliphatic side chain may contain within the chain up tothree of any combination of the heteroatoms O, S, or N, with the provisothat such heteroatoms cannot be adjacent to each other in the chain. Thehydroxylated aliphatic side chain may also have a carboxyalkylfunctionality.

Aryl denotes a mono or bi-cyclic aromatic system. Examples of preferredaryl groups include those having from 6 to 14 carbon atoms.Representative examples include phenyl, 1-naphthyl, 2-naphthyl, andindanyl. The aryl group may contain additional substituents selectedfrom the group consisting of: halogen atoms (e.g. Cl, Br, F and/or I),alkoxy, alkyl, and amino.

Alkylaryl denotes an aryl as described herein, wherein one of thehydrogens of the aryl is substituted by an alkyl as described herein.

A substituted phenyl refers to a phenyl bearing up to three substituentsindependently selected from the group consisting of --S--alkyl, --S--H,amino, NO₂, and O-alkyl; and up to five substituents independentlyselected from the group consisting of F, Cl, I, Br, alkyl, and OH.

A substituted benzyl refers to a benzyl with the phenyl portion thereofbearing up to three substituents independently selected from the groupconsisting of --S--alkyl, --S--H, amino, NO₂, and O-alkyl; and up tofive substituents independently selected from the group consisting of F,Cl, I, Br, alkyl, and OH.

As used herein, a boldfaced bond, denotes a bond which comes up out ofthe plane of the page. A dash bond, denotes a bond which comes downbelow of the plane of the page. A curved bond, denotes a racemicmixture.

Exemplary compounds of the invention include:

The invention relates to compounds of the formula

    __________________________________________________________________________     ##STR11##                                                                     ##STR12##                                                                        ##STR13##                                                                                          ##STR14##                                            __________________________________________________________________________        ##STR15##           CONH(CH.sub.2).sub.3 CH.sub.3                             ##STR16##           CONH(CH.sub.2).sub.7 CH.sub.3                             ##STR17##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR18##           CONH(CH.sub.2).sub.17 CH.sub.3                            ##STR19##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 1                  ##STR20##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 2                  ##STR21##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 1                  ##STR22##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 2                  ##STR23##           CONH(CH.sub.2).sub.13 CH.sub.3                        10.                                                                               ##STR24##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR25##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR26##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR27##           CONH(CH.sub.2).sub.9 CH.sub.3                             ##STR28##           CON(OBZL)(CH.sub.2).sub.11 CH.sub.3                       ##STR29##                                                                                          ##STR30##                                                ##STR31##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR32##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR33##           CON(OH)(CH.sub.2).sub.11 CH.sub.3                         ##STR34##                                                                                          ##STR35##                                            20                                                                                ##STR36##                                                                                          ##STR37##                                                ##STR38##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 1                  ##STR39##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer 2                  ##STR40##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR41##           CONH(CH.sub.2).sub.11 CH.sub.3                            ##STR42##           CONH(CH.sub.2).sub.11 CH.sub.3                        __________________________________________________________________________

In the above table and throughout the specification, OBZL denotesO-benzyl.

Preferred compounds of the invention are: ##STR43##

The most preferred compound of the invention is ##STR44##

The invention also relates to a pharmaceutical composition whichcomprises a compound of formula I in combination with a pharmaceuticallyacceptable carrier material,

The invention also relates to a method for treating fungi whichcomprises administering to a mammal in need of such treatment ananti-fungally effective amount of a compound of formula I for suchpurpose.

DETAILED DESCRIPTION OF THE INVENTION

Asymmetric centers exist in compounds of formula I of the invention.Accordingly, compounds of formula I include stereoisomers.

All such isomeric forms and mixtures thereof are within the scope of thepresent invention. Unless otherwise indicated, the methods ofpreparation disclosed herein may result in product distributions whichinclude all possible structural isomers, although it is understood thatphysiological response may vary according to stereochemical structure.The isomers may be separated by conventional means such as fractionalcrystallization or HPLC (high performance liquid chromatography).

The compounds of formula I can exist in unsolvated as well as solvatedforms, including hydrated forms, e,g. the hemihydrate. In general, thesolvated forms, with pharmaceutically acceptable solvents such as water,ethanol, and the like are equivalent to the unsolvated forms for thepurposes of the invention.

The compounds of formula I form pharmaceutically acceptable salts. Thepreferred pharmaceutically acceptable salts are nontoxic acid additionsalts formed by adding to a compound of the invention about astoichiometric amount of a mineral acid, such as HCl, HBr, H₂ SO₄ or H₃PO₄ or of an organic acid such as acetic, propionic, valeric, oleic,palmitic, stearic, lauric, benzoic, lactic, paratoluenesulfonic, methanesulfonic, citric, maleic, fumaric, succinic and the like, respectively,

The compounds of formula I above may be administered in compositionsthat also contain other anti-fungal agents. The compounds of formula Iabove may also be administered at the same time that other anti-fungalagents are being administered.

The compounds of formula I above may be prepared by the methodsdescribed below with reference to Schemes 1 and 2 wherein R₃, R₄, Het,J, K and Z are as described above unless otherwise indicated. ##STR45##

R₁₀ is an N-blocked natural amino acid, or an N-blocked metabolizablegroup; or a nitrogen protecting group such as tertbutoxycarbonyl orbenzyloxycarbonyl.

As used herein Osu denotes N-oxysuccinimide, HOsu denotesN-hydroxysuccinimide, DCC denotes 1,3-dicyclohexylcarbodiimide, HObtdenotes N-hydroxybenztriazole, DMF denotes N,N-dimethylformamide, THFdenotes tetrahydrofuran.

The compounds of formulas II and IV are known, can be prepared inaccordance with known methods, or else their preparation is describedherein.

A compound of formula II may be converted to the correspondingN-oxysuccinimide ester of formula III thereof by reaction withN-hydroxysuccinimide in an aprotic, organic solvent such asN,N-dimethylformamide, acetonitrile, ethyl acetate, methylene chloride,or more preferably, tetrahydrofuran in the presence of a carbodiimidesuch as 1,3-dicyclocarbodiimide, and at a temperature in the range ofabout 0° to about 40° C. or more preferably 20° C. The resulting esterof formula III may be isolated by conventional means such ascrystallization or chromatography, or it may be used directly in thenext step of a synthesis of this invention.

Procedure A

An N-oxysuccinimide ester of formula III may be reacted with a uracilpolyoxin C compound of formula IV ##STR46## wherein J, K, and Het are asdescribed above, to obtain a compound of formula V. The reaction is runin apolar, aprotic, organic solvent such as N,N-dimethylformamide,acetonitrile, tetrahydrofuran, or more preferably, dimethylsulfoxide.The reaction is run under an inert atmosphere such as argon or nitrogen.The reaction is run at a temperature in the range of about to 20° C. toabout 50° C., more preferably, at room temperature. The resultingcompound of formula V may be separated by conventional means such ascrystallization or chromatography, or it may be used directly in thenext step of a synthesis of this invention.

Procedure B

A compound of formula V may be reacted with an amine R₅ NR₆ (wherein R₅and R₆ are as described herein) to obtain a compound of formula VI. Thereaction is run in a polar, aprotic, organic solvent such asacetonitrile, tetrahydrofuran, or more preferably, dimethylformamide, inthe presence of a carbodiimide, such as dicyclohexylcarbodiimide, and inthe further presence of an activating reagent such asN-hydroxysuccinimide, or more preferably 1-hydroxybenztriazole. Thereaction is run at a temperature in the range of about 0° to about 50°C., more preferably, room temperature. The resulting compound of formulaVI may be separated by conventional means such as crystallization orchromatography, or it may be used directly in the next step of thesynthesis of this invention.

Amines of the formula R₅ NR₆ are known, or may be prepared in accordancewith known methods.

The next step of the synthesis is the deprotection of the --NHR₁₀ groupof a compound of formula VI to obtain a compound of formula I.

In the case of the tert-butoxycarbonyl (BOC) protecting group, acompound of formula VI may be convened to a final product of formula Iof the invention by treatment with a mineral acid such as hydrochloricacid or sulfuric acid, or more preferably, the organic acidtrifluoroacetic acid at a temperature in the range of about 0° C. toabout 25° C., more preferably more temperature. A compound of formula Iof the invention may be separated from reaction mixture by conventionalmeans such as crystallization or chromatography.

In the case of the benzyloxycarbonyl (CBZ) protecting group, a compoundof formula VI may be hydrogenolized by dissolving it in a solvent suchas ethanol, or more preferably, 95% methanol and 5% formic acid in thepresence of a catalyst which facilitates hydrogenolysis such aspalladium on carbon, or more preferably palladium black. Afterdeprotection is complete, the palladium may be filtered and the filtrateevaporated under reduced pressure at 50° C. to obtain a compound offormula I of the invention. This compound of formula I may be furtherpurified by conventional means such as crystallization orchromatography.

Compounds of formulas II and III are known, and may be prepared inaccordance with known methods, or else they may be prepared as describedherein. ##STR47## wherein. L is a protecting group or a leaving groupsuch as CBZ or BOC.

Procedure C

A compound of formula VII may be reacted with a protecting group or aleaving group such as benzyloxycarbonyl (CBZ) or, more preferably, BOC(di-tert. butyldicarbonate) to obtain a compound of formula VIII. Thesolvent employed is a polar solvent such as methanol,N,N-dimethyiformamide, or more preferably, water. The reaction is run ata pH in the range of about 8 to about 10, more preferably, 9.5. Thereaction mixture is kept basic through use of an alkali metal carbonatesuch as sodium carbonate or potassium carbonate, or through use of analkali metal hydroxide such as potassium hydroxide or, more preferably,sodium hydroxide. The reaction is run at a temperature in the range ofabout 0° to about 25° C., more preferably about room temperature.

The resulting compound of formula VIII may be separated by conventionalmeans such as crystallization or chromatography, or it may be useddirectly in the next step of the synthesis of this invention.

A compound of formula VIII may be reacted with an amine R₅ NR₆ asdescribed above, to obtain a compound of formula IX. The reaction is runin a polar, organic solvent such as acetonitrile, tetrahydrofuran, ormore preferably, dimethylformamide, in the presence of a carbodiimide,such as dicyclohexylcarbodiimide, and in the further presence of anactivating reagent such as N-hydroxysuccinimide, or more preferably1-hydroxybenztriazole. The reaction is run at a temperature in the rangeof about 0° to about 50° C., more preferably, room temperature. Theresulting compound of formula IX may be separated by conventional meanssuch as crystallization or chromatography, or it may be used directly inthe next step of the synthesis of this invention.

Procedure D

A compound of formula IX may be converted to a compound of formula X byreaction in a mineral acid such as hydrochloric acid or sulfuric acid,or more preferably, the organic acid trifluoroacetic acid. The reactionis run at a temperature in the range of about 0° to about 25° C., morepreferably room temperature. The resulting compound of formula X may beseparated by conventional means such as crystallization orchromatography, or it may be used directly in the next step of thesynthesis of this invention.

A compound of formula X may be convened to a compound of formula VI byreaction with a N-oxysuccinimide ester of a N-blocked-amino acid of theformula ##STR48## wherein R₁₀ is as described herein. The reaction isrun in an anhydrous polar organic solvent such as methyl sulfoxide,tetrahydrofuran, acetonitrile, or more preferably dimethylformamide, inthe presence of a tertiary amine base such as diisopropylethylamine,N-methyl morpholine, or more preferably triethylamine at a temperaturein the range of about 0° to about 50° C., or more preferably roomtemperature. The resulting compound of formula VI may be separated byconventional means such as crystallization or chromatography, or it maybe used directly in the next step of the synthesis of this invention.

The next step of the synthesis is the deprotection of the --NHR₁₀ groupof a compound of formula VI to obtain a compound of formula I.

In the case of the tert-butoxycarbonyl (BOC) protecting group, thecompound of formula VI may be converted to a final product of formula Iof the invention by treatment with a mineral acid such as hydrochloricacid or sulfuric acid, or more preferably, the organic acidtrifluoroacetic acid, at a temperature in the range of about 0° to about25° C., more preferably room temperature. The compound formula I of theinvention may be separated from the reaction mixture by conventionalmeans such as crystallization or chromatography.

In the case of the benzyloxycarbonyl (CBZ) protecting group, a compoundof formula VI may be hydrogenolized by dissolving it in a ethanol, ormore preferably, 95% methanol and 5% formic acid in the presence of acatalyst which facilitates hydrogenolysis such as palladium on carbon,or more preferably, palladium black. After deprotection is complete thepalladium may be filtered and the filtrate evaporated under reducedpressure at 50° C. to obtain a compound of formula I of the invention.This compound of formula I may be further purified by conventional meanssuch as crystallization or chromatography.

Compounds of formulas VII and XI are known, may be prepared inaccordance with known methods, or else they may be prepared as describedherein.

The compounds of formula I of the invention are useful as agents fortreating fungi. Compounds of formula I of the invention were tested andfound to be active as anti-fungal agents using procedures set forthbelow.

RESULTS

The minimum inhibitory concentrations (MIC-R and MIC-S) for a series ofcompounds of formula I of the invention are as shown in Table 1 below.Mass spectrum (MS) molecular ion masses are also shown in this table.

The test results shown below (MIC-R and MIC-S) were obtained from thetest procedures set forth just below.

In vitro anti-fungal activity was determined in microtiter minimuminhibitory concentration (MIC) tests using Yeast Nitrogen Broth (YNBwithout amino acids, Difco., Detroit, Mich.) at pH 5.4. Yeasts weregrown overnight in Sabouraud Dextrose Broth at 28° C. with shaking, andconcentrations adjusted in sterile saline using a spectrophoptometer at540 mμ. Compounds were dissolved in various vehicles and diluted inmedia to twice the final concentrations. The Cetus Pro/Pette system wasused to serially dilute 50 μl in round bottom 96 well plates (Falcon,Lincoln Park, N.J.). The turbidometrically adjusted yeast suspensionswere diluted 1:3,000 in YNB. These dilutions when added to the wells,produced a final inoculum of 3×10³ /ml. Plates were incubated at 37° C.for 48 hours. MICs were defined as the lowest concentrations of compoundthat prevented visible growth. MICs were taken against the followingorganisms and expressed in the table above as the geometric mean.

MIC-R and MIC-S mean respectively MICs taken against resistant andsensitive strains.

    ______________________________________                                                Resistant Strains                                                             Candida albicans C79                                                          Candida albicans C6                                                           Candida albicans C8                                                           Candida albicans C31                                                          Candida albicans C54                                                          Candida albicans C70                                                          Candida albicans C104                                                         Candida albicans C138                                                         Candida albicans C140                                                         Candida stellatoidea C45                                                      Sensitive Strains                                                             Candida albicans C141                                                         Candida sp. C2                                                                Candida sp. C109                                                              Candida parapsilosis C67                                                      Candida tropicalis C112                                                       Torulopsis glabrata C95                                                       Candida pseudotropicalis C967                                                 Candida guillermondii C137                                                    Sacchararomyces C78                                                           Sacchararomyces mutant C147                                           ______________________________________                                    

                                      TABLE 1                                     __________________________________________________________________________     ##STR49##                                                                     ##STR50##                                                                         ##STR51##                                                                                          ##STR52##                                                                                              ##STR53##                                                                          ##STR54##                                                                         ##STR55##         __________________________________________________________________________         ##STR56##           CONH(CH.sub.2).sub.3 CH.sub.3                                                                          >4096                                                                              >4096                                                                             544                     ##STR57##           CONH(CH.sub.2).sub.7 CH.sub.3                                                                          478  460 600                     ##STR58##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         23   478 657                     ##STR59##           CONH(CH.sub.2).sub.17 CH.sub.3                                                                         >2048                                                                              >2048                                                                             740                     ##STR60##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 >256 >256                                                                              620                     ##STR61##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 74   181 620                     ##STR62##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 17   103 680                     ##STR63##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 >256 158 680                     ##STR64##           CONH(CH.sub.2).sub.13 CH.sub.3                                                                         >256 147 684                10.                                                                                ##STR65##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         147  478 646                     ##STR66##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         316  85  659                     ##STR67##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         28   208 659                     ##STR68##           CONH(CH.sub.2).sub.9 CH.sub.3                                                                          181  388 628                     ##STR69##           CON(OBZL)(CH.sub.2).sub.11 CH.sub.3                                                                    28   119 762                     ##STR70##                                                                                          ##STR71##               143  362 692                     ##STR72##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         294  239 666                     ##STR73##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         21   158 666                     ##STR74##           CON(OH)(CH.sub.2).sub.11 CH.sub.3                                                                      39   239 672                     ##STR75##                                                                                          ##STR76##               111  194 700                20.                                                                                ##STR77##                                                                                          ##STR78##               >256 >256                                                                              578                     ##STR79##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 208  158 673                     ##STR80##           CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                                 >256 158 673                     ##STR81##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         9.8  56  631                     ##STR82##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         18   194 587                     ##STR83##           CONH(CH.sub.2).sub.11 CH.sub.3                                                                         >256 >256                                                                              654                __________________________________________________________________________

As can be seen from the above table, compounds of the invention exhibitanti-fungal activity against human and animal pathogens which are bothsensitive to nikkomycins (MIC-S μg/ml<128) and resistant to nikkomycins(MIC-R μg/ml>2048).

In vivo anti-fungal activity was tested for in the following testprotocol. Compounds of the invention were found to be active in thefollowing test protocol.

MATERIALS & METHODS

Vaginal Candida infections in hamsters.

C. albicans (C60 or C79), clinical isolates, were grown on SDA slantsfor 48 h at 28° C. Cells were washed off the Slants with SDB broth toobtain a suspension of approximately 1×10⁸ cells/ml. Groups of 10 femaleSyrian outbred hamsters (Charles River), weighing 100-120 grams, wereused. On the first day of the experiment, the vagina was swabbed with adry cotton swab to remove any mucus and to induce a slight irritation.The suspension of C. albicans (0.05 ml) was introduced into the vaginaon three successive days using a syringe equipped with a blunt needle.Two days after infection, samples were obtained for culture by insertinga sterile cotton swab into the vagina. The swabs were placed into 10 mlof 0.9% saline containing cycloheximide (Actidione, Upjohn, 0.45 g/l)and chloramphenicol (Chloromycetin sodium succinate, Parke Davis, MordsPlains, N.J., 0.1 g/l), and then vigorously agitated to dislodge thevaginal sample. A 2 ml aliquot of each sample was then passed through a0.45 micron Millipore filter. Following a saline rinse of the filters,the filters were placed onto mycosel agar plates and incubated at 37° C.After 48 h the number of C. albicans colonies on the filters werecounted. Only animals that showed positive cultures were used for theexperiments.

Treatment began 4 days after completion of infection. Compounds weresolubilized in ethanol:PEG400: glycerol (10:45:45). Treatment wasintravaginally at concentrations of 0.125-2% once daily for 4 days.Using cotton swabs, vaginal samples were obtained after 2, 4, 7 and 9days of treatment and the swabs were processed as above. Efficacy wasdetermined on the basis of negative cultures at each of the four timeperiods.

MATERIALS & METHODS

C. albicans infection studies in mice (PD₅₀).

C. albicans Wisconsin (C 43) and C. tropicalis (C 112), grown onSabouraud dextrose agar (SDA) slants for 48 h at 28° C., were suspendedin saline and adjusted to 46% transmission at 550 nm on aspectrophotometer. The inoculum was further adjusted by hemacytometerand confirmed by plate counts to be approximately 1 or 5×10⁷ CFU/ml.CF-1 mice (white, male, ca. 20 g, Harlan Sprague Dawley, Inc.,Indianapolis, Indiana) were infected by injection 1 or 5×10⁶ CFU intothe tail vein. Antifungal agents were administered intravenously orsubcutaneously in ethanol: water (10:90), 4 h post infection and oncedaily thereafter for 3 or 4 more days. Survival was monitored daily. Theprotective dose₅₀ (PD₅₀) was defined as that dose which allowed for 50%survival of mice.

The pharmaceutical compositions of the present invention may beformulated by combining a compound of the invention or pharmaceuticallyacceptable salt thereof with any suitable diluent, i.e., inertpharmaceutical carrier or diluent adapted for administration orally,parenterally, topically, vaginally or rectally.

Examples of suitable compositions include solid or liquid compositionsfor oral administration such as tablets, capsules, pills powders,granules, solutions, suspensions or emulsions. They may also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, physiological saline or some sterileinjectable medium immediately before use.

Topical dosage forms may be prepared according to procedures well knownin the art, and may contain a variety of ingredients, excipients, andadditives. The formulations for topical use include ointments, creams,lotions, powders, aerosols, pessaries, and sprays. Of these, lotions,ointments, and creams, may contain water, oils fats, waxes, polyesters,alcohols or polyols, plus such other ingredients as fragrances,emulsifiers, and preservatives. Powders are made by mixing the activeingredient with a readily available, inert, pulverous distributing agentsuch as talcum, calcium carbonate, tricalcium phosphate, or boric acid.Aqueous suspensions of the above powders may also be made. Solutions oremulsions may also be prepared using inert solvents which are preferablynonflammable, odorless, colorless, and nontoxic, for example, vegetableoils, isopropanol, dimethyl sulfoxide, hydrogenated naphthalenes, andalkylated naphthalenes. Similarly, aerosol, and non aerosol sprays maybe prepared using solutions or suspensions in appropriate solvents, forexample, difluorodichloromethane for aerosols.

Parenteral forms to be injected intravenously, intramusculady, orsubcutaneously, are usually in the form of a sterile solution, and maycontain salts or glucose to make the solution isotonic.

Compounds of the invention may also be incorporated in vaginal or rectalsuppositories. Suppositories may be prepared by methods which areconventional in the art. In addition to comprising a compound of theinvention, suppositories may contain a suppository base made up ofbiocompatible polymers, a surfactant, and an absorbent in a vegetableoil phase.

In addition, the suppositories may be further modified by inclusion ofan antioxidant.

When used orally or parenterally, the compounds of the invention can beadministered in an amount ranging from about 0.02 mg/kg body weight toabout 40.0 mg/kg body weight, preferably from about 0.1 mg/kg bodyweight to about 20 mg/kg body weight per day.

Determination of the proper dosage of a compound of the invention for aparticular situation is within the skill of the art. Generally,treatment is initiated with smaller dosages that are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired.

The amount and frequency of administration of the compounds of formula Iand the pharmaceutically acceptable salts thereof will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition, size of the patient, severity of the symptombeing treated, and the pharmacokinetics of the particular compound beingemployed.

The invention disclosed herein is exemplified by the followingpreparative examples, which should not be construed to limit the scopeof the disclosure. Alternative mechanistic pathways and analogousstructures within the scope of the invention may be apparent to thoseskilled in the art.

EXAMPLE 1 Preparation of N-BOC-UPOC ##STR84##

UPOC (8 g, 27.8 mmol) was dissolved in 200 ml of distilled water. Thesolution was stirred and the pH was adjusted to 9.5 with 20% sodiumhydroxide solution. At room temperature di-tert. butyldicarbonate (8 g,36.7 mmol) was added all at once and stirred while the pH was monitoredat 9.5 with sodium hydroxide. After approximately 5 hours or after thereaction was complete by thin layer chromatography (tlc) (40%methanol-methylene chloride, normal phase silica plates) DowexXFS-43279.00 hydrogen from resin was added to pH 3.0. The resin filteredoff and the solvents were evaporated off under reduces pressure at 45°C. The resulting solid was dissolved in 100 mL of methanol and added tostirring ether. The precipitate was filtered off and dried in a vacuumdesiccator to obtain 10.5 g (97%) of the title product.

¹ H-NMR (300 MHZ, CD₃ OD) δ7.70 (1H, d, J=7.5 Hz), δ5.92 (1H, d, J=6Hz), δ5.70 (1H, d, J=7.5 Hz), δ4.39 (1H, m,), δ4.32 (1H, m), δ4.23 (1H,m), δ4.12 (1H, t, J=5.25 Hz), δ1.45 (1H,s).

EXAMPLE 2 Preparation of N-BOC-6'-dodecylamido-UPOC ##STR85##

N-BOC-UPOC (5.2 g, 13.42 mmol) was dissolved in 150 ml of dryN,N-dimethylformamide (DMF). Dodecylamine (3.73 g, 20.13 mmol),dicyclohexylcarbodiimide (5.538 g, 26.84 mmol), and1-hydroxybenztriazole (2.26 g, 16.77 mmol) were added and the resultingmixture was stirred at room temperature. After 48 hours 2 ml of H₂ Owere added and the resulting mixture was stirred for 15 minutes. Theprecipitate was filtered off and the solvent, DMF, was evaporated underhigh vacuum at 50° C. to obtain an oil. The oil was chromatographed onsilica gel using 1.25% to 5% methanol-methylene chloride to obtain 6.3 g(84%) of the title product.

EXAMPLE 3 6'-Dodecylamido-UPOC-trifluoroacetate ##STR86##

N-BOC-6'-dodecylamido-UPOC (6.25 g, 11.25 mmol) were dissolved in 15 mlof trifluoroacetic acid and the resulting mixture was stirred at roomtemperature for 5 minutes. 100 ml of ether were added and the solidswere filtered off to obtain 6.01 g of title product after drying in avacuum desiccator.

¹ H-NMR (300 MHZ, D₂ O) δ7.62 (1H, d, J=8.07 Hz), 5.66 (1H, d, J=8.01Hz), 5.49 (1H, d, J=8.87 Hz) 4.28 to 4.34 (2H, m), 4.16 (1H, t, J=4.95Hz), 4.08 (1H, d, J=5.22 Hz), 1.46 (2H, m), 1.22 (20H, br.s), 0.835(3H,t, J=6.99 Hz).

EXAMPLE 4 Preparation of 5-N-(α-N-BOC-5-hydroxy-tryptophanyl)-UPOC##STR87##

To a mixture of UPOC (3.3 g, 11.4 mmol) in 350 ml of DMSO and 9 ml ofNMM were added the N-oxysuccinimide ester of L-5-hydroxytryptophan (7.6g, 17.5 mmol) and the resulting mixture was stirred at room temperatureunder a dry nitrogen atmosphere. After 24 hours 600 ml of ether wereadded and the resulting mixture was stirred for 5 minutes. The mixturewas allowed to settle and the supernatent ether-DMSO solution wasdecanted off. This sequence was repeated two more times to obtain agummy solid. The gummy solid was chromatographed on C-18 reverse phasesilica gel using 10 to 50% methanol/water gradient to obtain 2.34 g(35%) of title product. FABMS 590.2 (M⁺ +1).

EXAMPLE 5 Preparation of 6'-Dodecylamido-5-N-(5-hydroxytryptophanyl)UPOC##STR88##

5-N-(L-N-BOC-5-hydroxy-tryptophanyl)-UPOC (0.2 g, 0.4 mmol) weredissolved in 10 ml of DMF. Hydroxybenztriazole (0.081 g, 0.6 mmol) andDCC (0.12 g, 0.6 mmol) and n-dodecylamine (0.11 g, 0.6 mmol) were addedand the solution was stirred at room temperature. After 24 hours, 1 mlof water was added and the resulting mixture was evaporated to drynessunder vacuum at 50° C. The residue was chromatographed on silica gelusing a mixture of 10% methanol/methylene chloride as the eluent toobtain 0.107 g of pure blocked product. The blocked product wasdissolved in 1 ml of trifluoroacetic acid and allowed to stand for 5minutes. Ether was added and the pure title product 0.1 g (35%) wasfiltered off.

FABMS 657 (M⁺ +1), ACC. MASS for C₃₃ H₄₉ N₆ O₈ 657.3612 found 657,3613.

EXAMPLE 6 Preparation of2-N-fluorenylphenyl-3-methyl-4-(p-methoxyphenyl)-4-hydroxy-butyryllactone##STR89##

3-S-N-fluorenylphenyl-2-methyl-3-carboxymethyl-butyral [(see H. Rapaportand J. Wolf, J. Org. Chem. 1989, 54, 3164-3173 (1.38 g, 3.57 mmol)] wasdissolved in 5 ml of tetrahydrofuran (THF). The solution was cooled to0° under a dry nitrogen atmosphere. 7.14 mmol of 4-anisole Grignardreagent was added and the mixture was stirred for 4 hours. The reactionmixture was added to 50 ml of saturated ammonium chloride solution andthe product was extracted with three 50 ml portions of ethyl acetate.The ethyl acetate extracts were dried over magnesium sulfate, filtered,and evaporated to an oil. The oil was chromatographed on a silica gelcolumn using a mixture of 5% ethyl acetate/hexane as the eluent toobtain 0.58 g (36%) of title product in a 3/1 mixture of trans/cisisomers.

¹ H-NMR (300 MHZ, CDCL₃) δ6.6 to 7.8 (18H, m), 5.47 (d, J=7.23 Hz) and4.91 (d, J=7.2 Hz) total of 1H in a ratio of 3/1 respectively, 3.75 and2.86 (1H, two d, J=7.02 in a ratio of 3/1 respectively, 3.73 and 3.76(3H two s in a ratio of 3/1 respectively), 2.25 and 2.55 (1H, m), 0.66and -0.08 (1H, two d, J=7.11 in a ratio of 3/1 respectively.

EXAMPLE 7 Preparation of2-S-N-BOC-amino-3-R,S-methyl-4-P-methoxyphenyl-butyric acid ##STR90##

To2-N-fluorenylphenyl-3-R,S-methyl-4-(p-methoxyphenyl)-4-hydroxy-butyryllactone(0.48 g) were added 100 ml of methanol and 50 ml of 1N HCl and thestarting material was hydrogenated over a catalytic amount of 5%palladium on charcoal for 4 hours. The palladium on charcoal filteredoff and the pH was adjusted to 9.5 with 1N sodium hydroxide solution.0.3 g of di-tert.-butyl-dicarbonate was added and the resulting mixturewas stirred for one hour. The reaction mixture was added to an excess of5% citric acid and the product was extracted with three 50 ml portionsof methylene chloride. The methylene chloride layers were dried overmagnesium sulfate, filtered, and evaporated to obtain an oil. The oilwas chromatographed on a silica gel column using a mixture of 5%methanol/methylene chloride to obtain 0.16 g (68%) of the title product.

¹ H-NMR (300 MHZ, CDCL₃) δ7.10 (2H, d, J=8.49 Hz), 6.82 and 6.84 (2H,two d, J=8.49 Hz), 5.07 and 5.16 (1H, two d, J=8.55 and 9.48 Hzrespectively), 4.40 (1H, m), 2.74 (1H, m), 2.31 (2H, m) 1.46 (9H, s),0.86 (3H, m).

EXAMPLE 8 Preparation of5-N-(2-S-N-BOC-amino-3-R,S-methyl4-p-methoxyphenylbutyryl)-UPOC##STR91##

To a mixture of UPOC (0.287 g, 1 mmol) in 40 ml of DMSO and 0.8 ml ofNMM were added the N-oxysuccinimide ester of2-S-N-BOC-amino-3-methyl-4-p-methoxyphenyl-butyric acid (0.88 mmol) andthe resulting mixture was stirred at room temperature under a drynitrogen atmosphere. After 24 hours, 60 ml of ether were added and themixture was stirred for 5 minutes. The mixture was allowed to settle andthe supernatent ether-DMSO solution was decanted off. This sequence wasrepeated two more times to obtain a gummy solid. The gummy solid waschromatographed on C-18 reverse phase silica gel 10 to 50%methanol/water gradient to obtain 0.099 g (16%) of title product.

MS m/e (493.4, M⁺ +1).

EXAMPLE 9 Preparation of6'-Dodecylamido-5-N-(2-S-amino-3-R,S-methyl-4-p-methoxyphenyl-butyryl)UPOC ##STR92##

5-N-(2-S-N-BOC-amino-3-R,S-methyl4-p-methoxyphenyl-butyryl)-UPOC (0.099g, 0.16 mmol) were dissolved in 5 ml of dimethylformamide (DMF).Hydroxybenztriazole (0.032 g, 0.24 mmol) and DCC (0.05 g, 0.24 mmol) andn-dodecylamine (0.045 g, 0.24 mmol) were added and the solution wasstirred at room temperature. After 24 hours, 0.5 ml of water was addedand the mixture was evaporated to dryness under vacuum at 50° C. Theresulting mixture was chromatographed on silica gel using a mixture of10% methanol/methylene chloride as the eluent to obtain 0.06 g of pureblocked product. The blocked product was dissolved in 1 ml oftrifluoroacetic acid and allowed to stand for 5 minutes. Ether was addedand pure title product 0.051 g (42%) was collected by filtration.

ACC. MASS for C₃₄ H₅₄ N₅ O₈ 660.3972, found 660.3930.

EXAMPLE 10 Preparation of 1-chloro-2-(4-methoxypheny)-ethane ##STR93##

4-methoxy-phenethyl alcohol (15.2 g, 10 mmol) were dissolved in 200 mlof dichloromethane and at 0° C., 7.35 ml of thionyl chloride were added.The reaction mixture was allowed to warm to room temperature over 1/2hour. After stirring for 20 hours, the mixture was added to brine andextracted with dichloromethane. The dichloromethane was evaporated toobtain an oil which was chromatographed on silica gel using a mixture of30% ethyl acetate/hexane as the eluent to obtain 8.8 g (51%) of thetitle product.

¹ H-NMR (300 MHz, CDCl₃) δ7.17 (2H, d, J=8.5 Hz), 6.88 (2H, d, J=8.5Hz), 3.81 (3H,s), 3.69 (2H, t, J=7.68 Hz), 3.03 (2H, t, J=7.68 Hz)

EXAMPLE 11 Preparation of 2-L-S-N-BOC-amino-4-d-methoxyphenyl-butyricacid ##STR94##

Diethylformamidomalonate (15.2 g, 75 mmol) were dissolved in 100 ml ofDMF and the resulting mixture was cooled under a nitrogen atmosphere inan ice bath. A 60% oil dispersion of sodium hydride (3 gm) was addedportionwise and the resulting mixture was stirred for one hour.1-Chloro-2-(4-methoxypheny)-ethane (8.8 gm, 52 mmol) were dissolved inDMF and added to a stirring sodium diethylformamidomalonate mixture andheated to 80° C. After 4 hours the resulting mixture was added to brineand extracted with dichloromethane. The dichloromethane extracts weredried over magnesium sulfate and filtered. The solvent was evaporatedunder reduced pressure. The product was chromatographed on silica gelusing a mixture of 50% ethyl acetate/hexane to obtain 11.01 g ofproduct. 250 ml of 10% HCl were added and the mixture was refluxed for 2hours. The water and HCl were evaporated off. To the resulting solid wasadded 100 ml of water and 25 ml of methanol. The pH was adjusted to 10.0with 25% sodium hydroxide and 10 g of di-tert. butyldicarbonate wereadded The mixture was stirred for 1 hour. The mixture was added to 500ml of 5% citric acid and the resulting mixture was extracted with 3×200ml portions of dichloromethane. The extracts were dried over magnesiumsulfate, and filtered. The solvent was evaporated off under reducedpressure. The resulting solid was chromatographed on silica gel using amixture of 5% methanol/dichloromethane to obtain 6.71 g (43%) of thetitle product.

¹ H-NMR (300 MHz, CDCl₃) δ7.12 (2H, d, J=8.55 Hz), 6.83 (2H, d, J=8.55Hz), 5.04 (1H, d, J=7.8 Hz) 4.35 (1H, m), 3.79 (3H, s 2.67 (2H, t,J=7.92 Hz), 1.92-2.19 (2H, m), 1.46 (9H, s).

EXAMPLE 12 Preparation of3-[(diethoxyphosphino)oxy]-2-methoxyimino-3-methylpropanoate ##STR95##

To a solution of ethyl3-[(diethoxyphosphino)oxy]-2-methoxyiminopropanoate (30 g, 0.11 moleTetrahedron Lett., 1988, 29 3361) in dimethylsulfoxide (DMSO) (150 ml)at 5°-8° C. was added potassium t-butoxide (13.7 g, 0.12 mole)in smallportions. The mixture was heated at 60° C. for 2 hours, then cooled to5°-8° C. and methyl iodide (7.3 ml, 0.12 mole) was added. The mixturewas heated at 40° C. for 6 hours, poured into an ice-cold solution ofammonium chloride (1000 ml), and extracted with methyl isobutyl ether.The ethereal extracts were evaporated and the residue waschromatographed (silica gel, 3:7 EtoAc-hexane) to obtain 17 g (54%) ofthe title compound.

¹ H-NMR (200 MHz, CDCl₃) δ3.8 to 4.15 (7H, complex m), 4.02 (3H, s),1.20 to 1.60 (9H, complex m).

EXAMPLE 13 Preparation of Ethyl-2-methoxyimino-3-methyl-4-(2-quinolino)-3-butenoate ##STR96##

Ethyl-2-methoxyimino-3-methyl-3-ethylphosphonatepropionate (3.6 g, 12.2mmol) were dissolved in 57 ml of DMF at room temperature under a drynitrogen atmosphere. A 60% oil dispersion of sodium hydride (0.58 g,14.6 mmol) was added portionwise and the resulting mixture was stirredfor 1 hour. Solid 2-quinolinecarboxaldehyde (1.59 g, 12 mmol) was added.After stirring for 4 hours, the reaction mixture was added to 5% citricacid and the resulting mixture was extracted with 3×100 ml ofdichloromethane. The dichloromethane evaporated off and the resultingmixture was chromatographed on silica gel using a mixture of 10%ethylacetate/hexane to obtain 1.8 g (50%) of the title product.

FABMS 299.0 (M⁺ +1).

EXAMPLE 14 Preparation of2-R-S-N-BOC-amino-3-R,S-methyl-4-(2-quinolinyl)butyric acid ##STR97##

Ethyl-2-methoxyimino-3-R,S-methyl-4-(2-quinolino)-3-butenoate (1.8 g, 6mmol) was dissolved in 37 ml of methanol. 37 ml of 1N potassiumhydroxide were added and the resulting mixture stirred at roomtemperature. After hydrolysis of the ethyl ester was complete, atapproximately 3 hours, as evidenced by thin layer chromatography (tic)(20% methano/dichloromethane) 12.4 g of nickel/aluminum amalgam wereadded and the resulting mixture was stirred. The temperature was allowedto rise to about 50° C. After stirring about four hours, the reactionmixture was filtered on a celite pad and the pH of the filtrate wasadjusted to 11 with 6N hydrochloric add. The white precipitate wasfiltered off. 1.6 g of di-tert.-butyldicarbonate were added and thereaction mixture was stirred with the pH being adjusted to 9.5 asnecessary. After 1 hour the reaction was complete. The reaction mixturewas added to 400 ml of 5% citric add and extracted with 3×150 ml ofdichloromethane. The dichloromethane was evaporated off andchromatographed. The resulting oil was chromatographed on silica gelusing a mixture of 5% methanol/dichloromethane to obtain 1.68 g (81%) ofthe title product as an 8/2 mixture of diastereomers.

¹ H-NMR (300 MHz, CDCl₃) of the first diastereomer: δ8.34 (1H, d, J=8.52Hz), 8.06 (1H, d, J=8.55 Hz), 7.90 (1H, d, J=7.95 Hz), 7.81 (1H, t,J=8.32 Hz), 7.64, t, J=7.86 Hz), 7.49 (1H, t, J=8.4 Hz), 5.723 (1H, d,7.92 Hz), 4.30 (1H, dd, J=4.62 and 6.78 Hz), 3.163 (2H, m), 2.52 (1H,m),1.45 (9H, s), 1.14 (3H, d, J=6.78 Hz); second diastereomer 8.29 (1H, d,J=8.34 Hz), 8.19 (1H, d, J=8.52 Hz), 7.88 (1H, d, J=7.92 Hz), 7.80 (1H,t, J=7.11 Hz), 7.62 (1H, t, J=7.35 Hz), 7.41 (1H, d, J=8.40), 5.57 (1H,d, J=7.92 Hz), 4.31 (1H, t, J=7.95 Hz), 3.29 (2H, m), 2.49 (1H, m), 1.42(9H, s), 1.05 (3H, d, J=7.05 Hz). FABMS 345.0 (M⁺ +1).

EXAMPLE 15 Preparation ofMethyl-2-S-N-fluorenylphenylamino-3-R,S-methyl-4-bromo butyrate##STR98##

1 g of methyl-2-S-N-fluorenylphenyl-3-R,S-methyl-4-hydroxy butyrate(preparation as described in H. Rapoport and J. Wolf, J. Org. Chem. 54,3164 (1989)) was dissolved in tetrahydrofuran. Carbon tetrabromide (1.57g) and triphenylphosphine (1.26 g) was added and the mixture was stirredat room temperature for 4 hours. The mixture was added to brine andextracted with ethyl acetate. The ethyl acetate layer was added overmagnesium sulfate, filtered, and evaporated to dryness. The mixture waschromatographed on a silica gel column using a mixture of 5% ethylacetate/hexanes as the eluent to obtain 1.07 g of the title product.

¹ H-NMR (300 MHZ, CD₃ OD) δ1.06 (3H, d, J=6.8 Hz), 1.84-1.90 (1H, m),3.21 (2H, d, J=6.8 Hz), 3.28 (3H, s), 7.162-7.47 (13H,m). FABMS 450 (M⁺+1).

EXAMPLE 16 Preparation of2-S-N-fluorenylphenylamino-3-R,S-methyl-4-(1H-1,2,4-triazolyl)butyricacid ##STR99##

Methyl-2-S-N-fluorenylphenyl-3-R,S-methyl-4-bromobutyrate (1.34 g, 3mmol) were dissolved in 10 ml of N,N-dimethylformamide.Sodium-1,2,4-triazole (0.32 g, 3.5 mmol) was added and the mixture wasstirred at room temperature. After 18 hours the mixture was added tobrine and the product was extracted with methylene chloride. Themethylene chloride layer was dried with magnesium sulfate, filtered, andevaporated to dryness. The resulting mixture was chromatographed on asilica gel column eluting with 30% ethyl acetate/hexane to obtain 0.85 gof the title product as the methyl ester. The methyl ester was dissolvedin distilled water/dioxane (10 ml/20 ml) and 1 g of potassium hydroxidewas added. The mixture was heated to 80° C. for 45 minutes and acidifiedwith citric acid. The product was extracted into methylene chloride toobtain the title product, 0.82 g.

¹ H-NMR (300 MHZ, CD₃ OD) δ0.86 & 0.89 (3H, two sets of doublets, J=6.9Hz), 2.1-2.3 (1H, m), 3.83-3.97(2H, m), 4.3(1H, dd, J=13.98 & 8.34 Hz),7.16-7.43(13H, m)

EXAMPLE 17 Preparation of2-S-N-fluorenylphenylamino-3-R,S-methyl-4-(1H-imidazolyl)butyric acid##STR100##

The preparation of this compound was identical to the preparation of2-S-N-fluorenylphenyl-3-R,S-methyl-4-(1H-1,2,4-triazolyl)butyric acidexcept sodium imidazole was used instead of sodium triazole.

EXAMPLE 18 Preparation of2-[(benzyloxycarbonyl)amino]-4-(5-hydroxy-2-pyridyl)-3-R,S-methylbutanoicacid ##STR101## A.

Methoxyethoxymethyl chloride (MEM) (19 g, 153 mmol) was added to asolution of 5-hydroxy-2-pyridinecarboxaldehyde (18.4 g, 149 mmol) in 300ml DMF. NaH (6.3 g of 60% oil dispersion, 158 mmol) were added inportions while cooling the mixture to maintain 20°-30° C. The mixturewas stirred an additional 1 hour at 25° C. 5 ml water were added, mostof the solvent was evaporated, and water was added to the residue. Themixture was extracted with EtOAc. The extracts were dried over Na₂ SO₄,filtered, and evaporated to a residue. The residue was chromatographedon silica gel eluting with EtOAc-hexanes (35:65) to obtain5-[(2-methoxyethoxy)methoxy]-2-pyridinecarboxaldehyde: C₁₀ H₁₃ NO₄(211.22); ¹ H-NMR (CDCl₃) δ10.01 (s,1), 8.52 (d,1), 7.93 (d,1), 7.49(m,1), 5.39 (s,2), 3.80 (m,2), 3.53 (m,2), 3.32 (s,3).

B.

NaH (3.9 g of 60% oil dispersion, 98 mmol) was added in portions to asolution of triethylphosphonopropionate (22 g, 92 mmol) in 200 ml THF.The mixture was then added slowly to a stirring a solution of5-[(2-methoxyethoxy)methoxy]-2-pyridinecarboxaldehyde (18.5 g, 88 mmol)in 50 ml THF at 15°-25° C., and the resulting mixture was stirred 18hours at room temperature. The THF supernatant liquid was decanted, theresidue was dissolved in water, and 2N HCl was added to adjust the pH to4.0. The mixture was extracted with hexane. The extracts were combined,the THF supernatant was decanted and evaporated. The residue wasextracted with hexanes. The extracts were dried over Na₂ SO₄, filtered,and evaporated to leave ethyl3-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-2-methylpropenoate (23.9 gmixed with 1.6 g mineral oil, 93% yield), which was used without furtherpurification: C₁₅ H₂₁ NO₅ (295.34); ¹ H-NMR (CDCl₃) δ8.54 (d,1), 7.65(m,1), 7.55-7.2 (m,2), 5.34 (s,2), 4.29 (q,2), 3.87 (m,2), 3.59 (m,2),3.38 (s,3), 2.34 (d,3), 1.38 (t,3).

C.

The product of part B (24 g, 81 mmol) was dissolved in 200 ml EtOH andhydrogenated with 10% palladium-on-carbon catalyst (0.75 g) at 1.5atmospheres for 18 hours. The catalyst was removed by filtration and thefiltrate was evaporated to leave ethyl3-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-2-methylpropanoate as a gum.

The gum was dissolved in 150 ml dioxane and 150 ml 1N KOH was added. Themixture was stirred vigorously for 24 hours, poured into water, andextracted with Et₂ O. The aqueous solution was cooled to 0° C. and thepH was adjusted to 2.5 with 12N HCl. The mixture was suction-filtered toseparate the precipitate, and the filtrate was extracted with EtOAc. Theprecipitate was dissolved with the EtOAc extract. The extract was driedover Na₂ SO₄, filtered, and evaporated to leave3-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-2-methylpropanoic acid.

This product was dissolved in 100 ml THF, 200 ml 1M borane-THF solutionwere slowly added, then the solution was heated at reflux for 6 hours.The mixture was cooled to room temperature, 40 ml MeOH was addedcarefully, then 100 ml of 5% aq. K₂ CO₃ were added and the mixture wasstirred vigorously. The mixture was concentrated to 1/3 volume, waterwas added, and the mixture was extracted with EtOAc. The extract wasdried over Na₂ SO₄, filtered, and evaporated to a residue. The residuewas chromatographed on silica gel eluting with EtOAc-hexanes (3:1) toobtain 3-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-2-methylpropanol: C₁₃H₂₁ NO₄ (255.32); ¹ H-NMR (CDCl₃) δ8.30 (d,1), 7.35 (dd,1), 7.07 (d,1),5.28 (s,2), 3.82 (m,2), 3.55 (m,2), 3.39 (s,3), 2.80 (d,2), 2.12 (m,1),0.94 (d,3).

D.

DMSO (4.8 ml, 68 mmol) was added dropwise at -60° C. to a solution ofoxalyl chloride (2.8 ml, 32 mmol) in 75 ml CH₂ Cl₂. A solution of theproduct of part C (7.6 g, 28 mmol) in 15 ml CH₂ Cl₂ was then addeddropwise at -60° C. After 1 hour at -50° C., 14 ml Et₃ N was slowlyadded, and the mixture was stirred without heating for 4 hours. Et₂ Owas added, the mixture was suction-filtered, the filtrate was washedwith water, dried over Na₂ SO₄, filtered and evaporated to a residue.The residue was chromatographed on silica gel eluting with EtOAc toobtain3-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-2-methylpropionaldehyde (3.8g, 48% yield): C₁₃ H₁₉ NO₄ MS m/e 253.30; ¹ H-NMR (CDCl3) δ9.84 (s,1),8.33 (d,1), 7.33 (dd,1), 7.08 (d,2), 5.27 (s,2), 3.80 (m,2), 3.55 (m2,),3.35 (s,3), 3.2-2.6 (m,3), 1.18 (d,3).

E.

The product of part D (4.2 g, 17 mmol) was added to a solution ofammonium carbonate (2.75 g, 29 mmol), NaCN (1.0 g, 20 mmol) and 20 mlwater. The mixture was stirred and heated at 100° C. for 3 hours, then12N HCl (approx. 3.8 ml) was added to the hot mixture until the pH was2-3. The mixture was evaporated to dryness, and the residue wasextracted with hot methanol. The methanol was evaporated to leave aresidue of crude hydantoin. This residue was dissolved in 20 ml water,50 g of Ba(OH)₂.8 H₂ O were added, and the resulting mixture was heatedat 110° C. for 18 hours. 250 ml of hot water were added to the mixtureand CO₂ was bubbled in until precipitation was complete. The mixture wassuction-filtered while hot, the filtrate cooled, 12N H₂ SO₄ was added toadjust the pH 2.5 and the mixture was suction-filtered again. Thefiltrate was poured through an AG-50W-X8 (H⁺) ion exchange column (75meq), washed with water, then eluted with 1M NH₄ OH. The basic eluatewas evaporated to leave2-amino-4-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-3-methylbutanoic acid(3.3 g, 67% yield) as a mixture of 4 stereoisomers: C₁₄ H₂₂ N₂ O₅(298.34); ms (FAB) 299 (M⁺ +1).

F.

Et₃ N (6.5 ml, 47 mmol) was added to a solution of the product of part E(3.8 g, 11 mmol) in 75 ml DMF. Then N-(benzyloxycarbonyloxy)succinimide(3.75 g, 15 mmol) was added and the mixture was stirred at roomtemperature for 20 hours. Most of the solvent was evaporated, 5% aq.citric acid was added, and the mixture was extracted with EtOAc. Theextracts were tided over Na₂ SO₄, filtered, and evaporated to a residue.The residue was chromatographed on silica gel eluting with CH₂ Cl₂-EtOAc-MeOH-HOAc (300:40:10:1) to obtain 2 diastereomeric pairs ofenantiomers of2-[(benzyloxycarbonyl)amino]-4-[5-[(2-methoxyethoxy)methoxy]-2-pyridyl]-3-methylbutanoicacid (1.32 g, 27% yield of the first to elute, (±)-(R*,R*)); 1.48 g, 31%yield of the second to elute, (±)-(R*,S*)): C₂₂ H₂₈ N₂ O₇ MS m/e(432.48); ¹ H-NMR (indistinguishable, CDCl₃) δ8.4 (d,1), 7.7-7.0 (m,2),7.3 (s,5), 6.1 (d,1, NH), 5.3 (s,2), 4.3 (m,2), 3.8 (m,2), 3.5 (m,2),3.3 (s,3), 3.1-2.4 (m,3), 0.9 (d,3).

G.-1

2,4-dimethoxybenzene (1.49 ml, 3.7 mmol) was added to a solution of thefirst product of part F to elute (1.35 g, 3.1 mmol) in 24 ml CH₂ Cl₂.Then trifluoroacetic acid (2.4 ml, 31 mmol) was added and the mixturewas stirred for 60 hours. 50 ml xylenes were added and the mixture wasevaporated to dryness. The residue was chromatographed on silica geleluting with CH₂ Cl₂ -EtOAc-MeOH-HOAc (gradient from 300:40:10:1 to40:40:10:1) to obtain(±)-(R*,R*)-2-[(benzyloxycarbonyl)amino]-4-(5-hydroxy-2-pyridyl)-3-methylbutanoicacid (0.94 g, 93% yield): C₁₈ H₂₀ N₂ O₅ (344.37); ¹ H-NMR (DMSO-d₆)δ8.04 (d,1), 7.71 (d,1,NH), 7.39 (s,5), 7.10 (dd,1), 7.03 (d,1), 5.04(s,2), 4.00 (dd,1), 2.85-2.45 (m,2), 2.35 (m,1), 0.78 (d,3).

G.-2

The procedure of part G.-1 above was used but the second product of partF was substituted to obtain(±)-(R*,S*)-2-[(benzyloxycarbonyl)amino]-4-(5-hydroxy-2-pyridyl)-3-methylbutanoicacid (0.88 g, 86% yield): C₁₈ H₂₀ N₂ O₅ MS m/e (344.37); ¹ H-NMR(DMSO-d₆) δ8.05 (d,1), 7.50 (d,1,NH), 7.36 (s,5), 7.05 (dd,1), 6.96(d,1), 5.06 (s,2), 3.97 (dd,1), 2.68 (q,1), 2.48 (m,2), 0.88 (d,3).

The following table illustrates how compounds of formula I of theinvention may be made by using procedures analogous to those set forthabove.

    __________________________________________________________________________     ##STR102##                                                                    ##STR103##                                                                      ##STR104##                                                                                        ##STR105##                                                                                           ##STR106##                                                                                   ##STR107##       __________________________________________________________________________       ##STR108##         CONH(CH.sub.2).sub.3 CH.sub.3                                                                        Use LNBOC-5-hydroxy-                                                          Tryptophan in procedure A                                                     Use n-butylamine                                                              in procedure B A/B               2                                                                                ##STR109##         CONH(CH.sub.2).sub.7 CH.sub.3                                                                        Use LNBOC-5-hydroxy-                                                          tryptophan for procedure A                                                    Use n-octylamine                                                              in procedure B A/B               3                                                                                ##STR110##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Procedure given                  4                                                                                ##STR111##         CONH(CH.sub.2).sub.17 CH.sub.3                                                                       Use LNBOC-5-hydroxy-                                                          tryptophan for procedure A                                                    use n-octadecylamine in                                                       procedure B    A/B               5                                                                                ##STR112##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 2-SNFluorinylphenyl-                                                      amino3-methyl-4-(1H-1,2,4-                                                    triazolyl)-butyric acid in                                                    procedure D    C/D               6                                                                                ##STR113##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 2-SNfluorinylphenyl-                                                      amino3-methyl-4-(1H-1,2,4-                                                    triazolyl)-butyric acid in                                                    procedure D    C/D               7                                                                                ##STR114##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 2-NBOC-amino-3-methyl-                                                    4-(2-quinolinyl)-butyric                                                      acid in procedure                                                                            C/D               8                                                                                ##STR115##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 2-NBOC-amino-3-methyl-                                                    4-(2-quinolinyl)-butyric                                                      acid in procedure                                                                            C/D               9                                                                                ##STR116##         CONH(CH.sub.2).sub.13 CH.sub.3                                                                       Use LNBOC-5-hydroxy-                                                          tryptophan in procedure A.                                                    Use n-tetradecylamine in                                                      procedure B    A/B               10                                                                               ##STR117##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use 2-SNBOC-amino-3-                                                          methyl-4-(5-hydroxypyridyl)-                                                  utyric acid in procedure                                                                     C/D               11                                                                               ##STR118##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use 2-SNfluorenylphenyl-                                                      amino3-methyl-4-(1H-                                                          imidazolyl)-butyric acid in                                                   procedure D    C/D               12                                                                               ##STR119##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use 2-SNBOC-amino-3-                                                          methyl-4-p-methoxyphenyl-                                                     butyric acid in procedure                                                                    C/D               13                                                                               ##STR120##         CONH(CH.sub.2).sub.9 CH.sub.3                                                                        Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure A                                                     Use n-decylamine                                                              in procedure B A/B               14                                                                               ##STR121##         CON(OBZL)(CH.sub.2).sub.11 CH.sub.3                                                                  Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure A.                                                    Use benzyloxy-n-dodecylamine                                                  n procedure B  A/B               15                                                                               ##STR122##                                                                                        ##STR123##            Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure C.                                                    Use farnesylamine in                                                          procedure B    A/B               16                                                                               ##STR124##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use 2-NBOC-amino-4-(2-                                                        quinolinyl)-butyric acid in                                                   procedure D    C/D               17                                                                               ##STR125##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use 2-NBOC-amino-4-(2-                                                        quinolinyl)-butyric acid in                                                   procedure D    C/D               18                                                                               ##STR126##         CON(OH)(CH.sub.2).sub.11 CH.sub.3                                                                    Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure C.                                                    Hydrogenolyze compound No.                                                    14 above.      A/B               19                                                                               ##STR127##                                                                                        ##STR128##            Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure C.                                                    Use2-amino-methyl-laurate in                                                  rocedure B.    A/B               20                                                                               ##STR129##                                                                                        ##STR130##            Use LNBOC-5-Hydroxy-                                                          tryptophan in procedure C.                                                    Use benzylamine in procedure                                                  B              A/B               21                                                                               ##STR131##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 5-hydroxy-3-(2-NBOC-                                                      amino-propionyl)- benzthiophe                                                 ne in procedure                                                                              C/D               22                                                                               ##STR132##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer                                                               Use 5-hydroxy-3-(2-NBOC-                                                      amino-propionyl)- benzthiophe                                                 ne in procedure                                                                              C/D               23                                                                               ##STR133##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use Lmethyl-NBOC-tyrosine in                                                  procedure D    C/D               24                                                                               ##STR134##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use NBOC-Lphenylglycine in                                                    procedure D    C/D               25                                                                               ##STR135##         CONH(CH.sub.2).sub.11 CH.sub.3                                                                       Use Nmethyl-NBOC-L tryptophan                                                  in procedure                                                                                C/D               __________________________________________________________________________

As used in the above table, OBZ means O-benzyl

What is claimed is:
 1. A compound of the formula: ##STR136## orpharmaceutically acceptable salts thereof, wherein Het is ##STR137## Ris H, COOH; C₁ -C₁₂ alkyl; CHO; CN; CH₂ OH; or CONH₂ ;wherein R₃ is##STR138## wherein R₈ is ##STR139## R₂ is H; OH; F; C₁ -C₆ alkoxy;alkyl; SH; S-alkyl; or SO₂ -alkyl; R₄ is H; a natural amino acidattached by a peptide bond; or a metabolizable group; R_(x) is C₁ -C₁₂alkyl; J is OH, H, Br, Cl, or F; K is OH, H, Br, Cl, or F; X and Y arethe same or different and are independently selected from the groupconsisting of H; OH; O--C₁ -C₁₄ alkyl; F; Cl; Br; NO₂, and alkyl; Z isR₅ NR₆ ; ##STR140## wherein Z' is R₅ NR₆, R₅ is H, a saturated orunsaturated C₆ -C₁₈ aliphatic side chain; or a hydroxylated C₆ -C₁₈aliphatic side chain; R₆ is H; OH; O-benzyl; O-aryl; O--C₄ -C₁₄ alkyl;C₁ -C₁₂ alkyl; phenyl; substituted phenyl; or CO--R₇ ; and R₇ is H, C₁-C₁₆ alkyl, aryl or alkylaryl; and n is an integer from 0 to
 16. 2. Acompound according to claim 1 wherein Het is uracil.
 3. A compoundaccording to claim 1 wherein R₄ is H.
 4. A compound according to claim3, wherein R₅ is (CH₂)₁₁ CH₃.
 5. A compound according to claim 1,wherein J and K are both OH.
 6. A pharmaceutical composition comprisinga compound according to claim 1 in combination with a pharmaceuticallyacceptable carrier.
 7. A method for treating a fungal infection in amammal which comprises administering to the mammal an anti-fungallyeffective amount of a compound according to claim
 1. 8. A compoundaccording to claim 1, selected from the group consisting of

    ______________________________________                                         ##STR141##                                                                    ##STR142##                                                                           ##STR143##                                                                                       ##STR144##                                         ______________________________________                                               ##STR145##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer 1                   ##STR146##         CONH(CH.sub.2).sub.11 CH.sub.3  isomer 2                   ##STR147##         CONH(CH.sub.2).sub.11 CH.sub.3                      ______________________________________                                    

or a pharmaceutically acceptable salt of such a compound.